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DISCUSSION

McInnes: When is Nrl turned on developmentally?

Swaroop: By RT-PCR we can detect it around E16.5 in embryonic mouse retina, but by Northern analysis it is more like E18.5. The antibodies we have currently pick up another protein called p45, which is present in all developing neurons. We are currently generating additional, more speci¢c antibodies.

McInnes: Is p45 a product of the Nrl gene?

Swaroop: No, it is encoded by a di¡erent gene expressed probably in all neural cells. It is antigenically similar.

Hauswirth: In the Nrl knockout mouse, is the enhanced photopic ERG amplitude due to the presence of more cones? Or is there a higher response from the cones that are there?

Swaroop: The ERG studies show a higher response but there is more S opsin. The outer segments of Cods (cone^rod hybrids) that are there have S opsin.

Hauswirth: So is the extra amplitude coming from ‘Cods’, not from a conversion of rods to real cones?

Swaroop: We don’t know what these Cods are. We use this term because we don’t want to call them cones. It is too early to say whether they are rods converted to cones. We are working on it.

Hauswirth: What about the rest of the phototransduction cycle in cones? Swaroop:It is all present in these Cod outer segments. All rod-speci¢c genes have

been switched o¡. None of the rod-speci¢c proteins are expressed, whereas every cone-speci¢c phototransduction protein that we have looked at is expressed at high levels.

Hauswirth: So if you want to preserve cone function in humans you just need to knock out NRL!

Bok: Anand Swaroop, did you say that the photoreceptors in this knockout mouse do not die?

Swaroop: The function of these cones is bizarre, and we do not see any large-scale change in the thickness of the outer nuclear layer at least for 6 months. So there is minimal cell loss during this period.

Bok: I presume the reason that Ed Stone and others looked at S-cone enhanced syndrome was because there is some sort of disease process in those retinas. Is there a cell loss, or is it just bizarre physiology?

Swaroop: I think there may be cell death in the rd7 mouse.

Farber: The rd7 mouse has a mutation in the PNR/Nr2e3 gene. NR2E3 mutations in humans cause enhanced S-cone syndrome (ESCS).

Bok: Do those humans lose cells?

Farber: No.

Bird: They have a restricted form of retinitis pigmentosa, and di¡erent mutations in the same gene cause a very severe form of retinal dystrophy.

Swaroop: My understanding is that there is some degeneration of photoreceptors in ESCS.

Dryja: They are di¡erent mutations. These are knockout mice, and all the humans are dominant missense mutations.

Bok: So are you talking about a gain of function in humans?

Swaroop: No. The human mutations in NR2E3 are also loss of function. Why do NR2E3 mutations lead to retinal degeneration, whereas we don’t see this in the Nrl knockout mice? We have only looked up to six months. The mice are now two years old and we are working with Dr Paul Sieving to examine the retina of older mice by histology and ERG to ¢gure out whether there has been any loss of cones. We have done some histology in mice older than 6 months and the outer nuclear layer is thinner. I don’t know whether there is slow loss of cone function, but we are working on it. I must state that these may not be real cones because they do express some rod markers. Our collaborator, Dr David Hicks, has two antibodies, Ret P3 and L1, which appear to speci¢cally recognize rods, not cones. These two antibodies recognize antigens in the knockout retina. In addition, Dr Enrica Strettoi has observed that the synaptic connections these Cods are forming are also apparently di¡erent from the normal rod and cone connections. According to Dr Ed Pugh, the Cods function as cones.

Farber: Many years ago we worked a lot with ground squirrels, and found that they had some cells that were intermediate between rods and cones. They all happened to be S cone cells. It might be worth looking here.

Swaroop: Maybe they don’t have NRL, and that is why they are all S cones. Zack: Have you used arrays on the Rd7 mouse to complement these?

Swaroop: Yes. We have done two time points but the data have not been analysed yet.

Bhattacharya: I have a general question about the microarray data. What is your feeling about the level of variability seen from one experiment to another?

Swaroop: The correlation coe⁄cient we get with A¡ymetrix GeneChips is over 0.99. If the same person dissects the retina and at the same time of the day the variability is minimum. If you take another knockout mice you see a little more variability. In slide microarrays we get closer to 0.98, so there is a little bit more variability in these. Even in slide microarrays there are ways to normalize the data. We are working with data-driven normalization. Rather than a global normalization of signals over the whole slide we do this on the basis of the data on each slide. This helps a great deal.

Aguirre: In an earlier talk Donald Zack showed a variability that was mainly patient related rather than age related. What are the prospects for looking at microarray data on patients?

Swaroop: I was talking primarily about mouse, where the data are very clean. We have done human studies with eight A¡ymetrix chips for young and eight A¡ymetrix chips with old retina, and there is a huge amount of variability within

the samples. You have to throw away many of the data that may be real but we can’t be con¢dent. I tend to be very conservative. This variability could be because of inherent variations in humans or because of many other factors, including tissue collection time and tissue preservation.

Cremers: There was a recent paper in Science showing the variance of di¡erent genes (Yan et al 2002). They showed that in families expression levels could vary in normal individuals twoto fourfold.

Swaroop: That is why we chose to work in mice. Cremers: Why do you think it is di¡erent in mice?

Swaroop: Because we are working with isogenic strains and we are controlling the sample preparation more carefully. With mice the variation is very low if the same strains are used. In humans there are many confounding factors.

Thompson: Are you using gender matching in your analysis?

Swaroop: Yes. My feeling is that there will be some genes that will show large variation, but most of the genes do not change. Once we de¢ne the baseline expression pro¢le of all genes it will be easier.

Bok: You would do a service to all of us to ¢gure out what the gender di¡erences are so we can subtract these out.

Swaroop: We are working on many aspects of microarray data with respect to retinal biology. We are also working on the development of databases and webbased sharing of information. We hope to make all of our data available on our website.

Bhattacharya: If we identify a mutation in a novel gene in humans, we may want to look at the disease biology (the impact of the mutation and how it might lead to cell death) through microarray techniques. If there is a huge amount of variation, would it be worthwhile generating a mouse model for each human gene and then studying the disease biology in the mouse?

Swaroop: From my own experience, the human work with microarrays has been very frustrating. Dr Shigeo Yoshida, a Japanese postdoc in my lab, had worked 16 h days for two years and produced few useful data, ultimately. For the postdoc’s sake it might be better to use mice than humans, at least initially. I would also advise people not to do just one time point: I would look at the progression of disease in the mouse model and pick four or ¢ve time points. It is a lot of work, but the information gained is very valuable.

Thompson: In terms of the human data, once we can get a line on more pathways a¡ected in retinal degeneration, so we are not looking at everything but just focusing on one pathway at a time, then it will be easier to see important changes.

Swaroop: Once you have de¢ned the pathways then you can ¢t in the data you get from human studies. It is much easier.

Zack: In terms of variability in di¡erent genes, I agree. In our experiments it turns out that rhodopsin is one of the most variable genes in the retina. Because

rhodopsin is expressed at such a high level it is very easy to measure accurately on an array, but the level can vary by sixfold just at the RNA level in age-matched individuals.

Bird: Does this vary by time of day?

Zack: We have too few data to answer that. But in mouse models rhodopsin is not one of the genes that is subject to signi¢cant circadian regulation.

Swaroop: That is a good point. With all our mice we dissect them between 12 and 2 pm because we are not sure whether this is a signi¢cant factor or not. I think it probably does matter. As long as everything is kept the same the variation is lower.

Reference

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